Generation of liver organoids by 3D co-culturing of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells

Abstract

Background and Hypothesis: Recent progress with combination of chemicals in cell culture media prolongs hepatocyte (HC) function in vitro. However, without other cells of hepatic lineage that comprise natural liver, HC alone are not suitable for the study of complex liver diseases. Hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) play vital roles in physiological and pathological liver function in situ. We hypothesized that coculturing primary HC with HSC and LSEC as 3D liver organoids in the same chemical conditioned media would uphold HC function over time, creating a better physiological 3D liver microenvironment for liver disease research.

Experimental Design: Freshly thawed primary human HCs were combined with immortalized human HSC alone, or together with immortalized human LSECs, to generate 3D liver spheroids. Spheroids were formed in HC maintenance media (HMM, Lonza) using low adhesion 96-well plates for 6 days before switching to the media with different combination of chemicals (SB31542, Forskolin, IWP2, DAPT, and LDN193189) for culturing another 14 days. Spheroids characterization, albumin secretion, mRNA transcription (CYP3A4), and histological analysis were performed for HC differentiation, maturation, and function.

Results: Both co-cultures of HC:HSC (2.5:1 ratio), and HC:HSC:LSEC (2.5:1:1) formed spheroids in HMM within 4 to 6 days (Fig.1A). The introduction of the different chemical-based media affected the roundness and diameter of spheroids differently (Fig.1B). In the presence of all 5 chemicals (5C), HC function was better maintained up to 21 days in HC:HSC:LSEC spheroids, measured by albumin, CYP3A4, and CK-19 secretion (Fig.1C). Surprisingly, 5C-based media significantly upregulated the expression of CK-19, which is one of the markers for cholangiocytes and liver precursor cells.

Conclusion and Potential Impact: 3D liver organoids composed of HC, HSC, and LSEC would create a niche environment mimicking the in vivo condition. Optimization of complex hepatic spheroids, as well as optimization of complex hepatic spheroid culturing media, would allow for the generation of a unique model for studying complex liver diseases.